Food Hydrocolloids 17 (2003) 211220 www.elsevier.

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Stability and rheology of emulsions containing sodium caseinate: combined effects of ionic calcium and non-ionic surfactant
Eric Dickinsona,*, Stewart J. Radforda, Matt Goldingb
a b

Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Unilever Research, Colworth Laboratory, Sharnbrook, Bedford MK44 1LQ, UK Received 19 March 2002; revised 15 May 2002; accepted 22 May 2002

Abstract We have investigated the effect of the combination of ionic calcium and non-ionic surfactant (Tween 20) on the visual creaming behaviour and rheology of n-tetradecane-in-water emulsions (4 wt% protein, 30 vol% oil, mean droplet diameter 0.4 mm) prepared at pH 6.8 with sodium caseinate. Varying concentrations of ionic calcium, expressed as the calcium/caseinate molar ratio R, were incorporated before homogenization, and varying concentrations of Tween 20 were added soon after homogenization. A stability diagram was constructed based primarily on whether the phase separation occurred after 48 h. Two distinct regions of enhanced emulsion stability could be identied. Shear rheology measurements indicated that most of the samples have a shear-thinning power-law-type behaviour. Addition of CaCl2 and/or Tween 20 resulted in a reduction in viscosity and a closer approach to Newtonian ow. Conrmation of the states of aggregation of the different emulsion compositions was obtained through confocal laser scanning microscopy and particle-size distributions from light scattering. Depletion occulation may be induced by excess caseinate, excess surfactant, or a combination of both, and is inuenced by interaction of the ionic calcium with the protein. Global stability behaviour can be understood in terms of the effect of ionic calcium on the nature of the adsorbed caseinate layer around the droplets and the state of aggregation of the non-adsorbed caseinate, and on the ability of Tween 20 to displace caseinate from the emulsion droplet surface. At low R, displacement from the interface of calcium-bound protein provides an extra source of (mainly protein-bound) ionic calcium in the aqueous phase, leading to fewer and larger caseinate aggregates that are then incapable of inducing depletion occulation. At high R, displacement of protein from the oil water interface results in emulsion restabilization through the disruption of calcium-induced caseinate bridges between the droplets. q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Caseinate; Calcium ions; Creaming; Depletion occulation; Rheology; Tween 20

1. Introduction Sodium caseinate, a soluble mixture of several different caseins (as1, as2, b and k), is widely used as an ingredient in the food industry. The surface-active caseins contain hydrophilic and hydrophobic groups in various sequences and proportions. Together in sodium caseinate they adsorb rapidly at the oil water interface during emulsication, thereby providing long-term stability to oil-in-water emulsions due to a combination of electrostatic and steric stabilization (Dickinson, 1997, 1999). However, for sets of ne emulsions of constant volume fraction and constant average droplet size, it has been established in practice that there is a nite range of caseinate concentration offering
* Corresponding author. Tel.: 44-1132-332956; fax: 44-1132332982. E-mail address: e.dickinson@leeds.ac.uk (E. Dickinson).

stability with respect to creaming and occulation (Dickinson & Golding, 1997a; Dickinson, Golding, & Povey, 1997). That is, at low caseinate/oil ratios, there is insufcient protein present to saturate fully the oil water interface during emulsication, and so the resulting emulsion is unstable to bridging occulation. Conversely, at high caseinate/oil ratios, the presence of excess (nonadsorbed) protein in the form of small caseinate aggregates (sub-micelles) may lead to a deterioration in creaming stability caused by depletion occulation. What gives the optimum stability, then, is an intermediate concentration of caseinate, enough to allow saturation coverage of the oil water interface, but without any signicant excess of protein remaining unadsorbed in the aqueous phase after emulsication (Dickinson, 1999). Factors affecting depletion occulation have been systematically studied in various simple model

0268-005X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 8 - 0 0 5 X ( 0 2 ) 0 0 0 5 5 - 3

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caseinate-based emulsions (Dickinson & Golding, 1997a, 1997b, 1998a; Dickinson et al., 1997; Srinivasan, Singh, & Munro, 2000, 2001; Ye & Singh, 2001), in caseinate-based emulsions with added non-adsorbing hydrocolloids (Cao, Dickinson, & Wedlock, 1990; Hemar, Tamehana, Munro, & Singh, 2001), and also in caseinate-based emulsions with added small-molecule surfactants (Dickinson & Ritzoulis, 2000; Dickinson, Ritzoulis, & Povey, 1999). Depending on the precise system composition, it appears that reversible occulation of droplets may be induced by non-adsorbed polysaccharides, by excess protein in the form of caseinate sub-micelles, or by excess emulsier in the form of surfactant micelles. Acceptable values of the depletion interaction free energy DGDEP of the order of a few kT can be calculated (Dickinson & Golding, 1997a; McClements, 1994) by putting reasonable numerical values of the parameters into the equation
2 DGDEP 22prm Prd 2 2rm =3;

where P is the osmotic pressure of the solution of submicelles, represented as a uid of hard spheres of radius rm, and rd is the mean droplet radius. It turns out that the susceptibility of a caseinate-stabilized emulsion to depletion occulation is sensitive to the concentration of electrolytes in the aqueous phase (Dickinson & Golding, 1998a; Srinivasan et al., 2000, 2001) as well as to the presence of other solutes such as ethanol (Dickinson & Golding, 1998b). In particular, it has been demonstrated (Dickinson & Golding, 1998a) that the addition of a moderate concentration of calcium ions (5 8 mM) can inhibit depletion occulation completely in a concentrated caseinate-stabilized emulsion system. Explaining this behaviour is not straightforward because ionic calcium can affect the relative proportions and the properties of the adsorbed and unadsorbed proteins according to various mechanisms. At the molecular level of interpretation, the calcium ions specically bind to phosphoserine residues on the caseins (Swaisgood, 1982), thereby modifying the molecular charge distribution, self-assembly behaviour and adsorption properties (Horne, 1998). In colloid science terms, the addition of divalent counter-ions like Ca2 has a large potential inuence on the structure of the electrical double-layer and hence on the range of electrostatic interactions between protein layers on different droplets (Dickinson, 1992). As calcium ions bind to the phosphoserine residues on the protein, the adsorbed caseins undergo conformational changes, which has the effect of reducing the adsorbed layer thickness and so diminishing the effectiveness of the steric stabilization mechanism (Horne, 1998). Furthermore, in some emulsions the caseinate-coated droplets can exhibit calcium-induced bridging occulation (Agboola & Dalgleish, 1995; Dickinson & Davies, 1999). In the case of emulsions containing an excess of nonadsorbed caseinate, it is suggested (Dickinson & Golding, 1998a) that the addition of calcium ions leads to a growth in

the average size of sub-micellar casein aggregates in the aqueous phase, and possibly also to some redistribution of non-adsorbed casein into the adsorbed layer. Taken together, these effects are predicted to cause a substantial reduction in the number density of caseinate sub-micelles, i.e. in the concentration of the small particles that are assumed to be the main depleting species responsible for inducing reversible occulation in the calcium-free systems. According to this argument (Dickinson & Golding, 1998a), on addition of 5 8 mM Ca2, the substantial reduction in osmotic pressure P makes the magnitude of DGDEP predicted from Eq. (1) too small to cause depletion occulation. Recently, we reported (Dickinson et al., 2001) a more rened analysis of the inuence of ionic calcium on the depletion occulation of caseinate-stabilized emulsions. This was based on the use of new thermodynamic data for caseinate solutions derived experimentally from combined static and dynamic light scattering. Successive thermodynamic approximations, excluding or including the effect of correlations between caseinate sub-micelles (i.e. in the absence or presence of the contribution from the second virial coefcient), were found to be in reasonable agreement with experiment. In particular, the theory predicts a pronounced decrease in depletion attraction strength at concentrations of Ca2 in the range 4 8 mM. Moreover, the allowance for interactive correlations was found (Dickinson et al., 2001) to provide an even better qualitative representation of emulsion occulation over the whole range of ionic calcium levels previously studied (2 14 mM) (Dickinson & Golding, 1998a). This rened analysis shows that the inferred decrease in depletion interaction strength is clearly consistent with marked changes in average molecular weight and sizes of protein aggregates with changes in Ca2 content, and also to more positive values of the second virial coefcient of caseinate sub-micelles. In the set of caseinate-based emulsions examined in the present study, the occulation state is inuenced separately by the concentrations of ionic calcium and non-ionic surfactant (Tween 20). Competitive displacement of adsorbed casein from the surface of emulsion droplets and from planar oil water interfaces has been well demonstrated in the literature (Chen, Dickinson, & Iveson, 1993; Courthaudon, Dickinson, & Dalgleish, 1991; Dickinson & Tanai, 1992; Mackie, Gunning, Wilde, & Morris, 2000). This competition has to be considered when interpreting the occulation behaviour of emulsions containing a mixture of sodium caseinate non-ionic surfactant, as summarized in a global stability diagram for the caseinate Tween 20 system by Dickinson et al. (1999). Motivated by observations of the sensitivity of emulsion occulation to the Ca2 content in surfactant-free systems (Dickinson & Golding, 1998a), the aim of the present work is to explore the role of ionic calcium content as an additional variable controlling depletion-induced occulation in caseinate Tween 20 emulsion systems. Construction of the global

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stability diagram is based on a combination of techniques, including rheology, particle-size distributions, visual creaming observations, and confocal microscopy. Throughout this paper, we adopt the term sub-micelle to denote the small self-assembled aggregates formed from sodium caseinate in the presence or absence of ionic calcium. Except at high Ca2 content, these caseinate submicelles are considerably smaller and less strongly associated than the native casein micelles found in skim milk, which are held together by colloidal calcium phosphate. There is no assumption here, explicit or implicit, that these caseinate sub-micelles are identicalstructurally or compositionallyto the putative sub-micelles present as building blocks in the well-known sub-unit model of the casein micelle.

2.3. Emulsion creaming Gravity creaming of emulsion samples was monitored visually after a period of 48 h using a method similar to that of Dickinson et al. (1999). Duplicate samples of emulsions of known composition (i.e. ionic calcium and Tween 20 concentrations) were stored in 75 mm 12 mm sample tubes (York Glassware, UK) for 48 h at 25 ^ 1 8C. Samples that exhibited signicant discernible creaming or phase separation after this time were deemed unstable. A sample was described as stable if it appeared homogeneous to the naked eye following this storage period. Stabilities of emulsion compositions containing high concentrations of calcium ions were characterized by their Mastersizer particle-size distributions. A bimodal particle-size distribution was taken to be indicative of non-reversible occulation, and a monomodal distribution (with no associated phase separation apparent in the creaming tube) to be indicative of a stable emulsion. 2.4. Emulsion rheology Steady-state viscometry measurements were carried out as a function of shear stress using a Bohlin CVO controlled stress rheometer. A double gap 40/50 measurement cell was used with a sample volume of 80 ml. The temperature was maintained at 25 ^ 1 8C. Measurements were made within 10 min of emulsion sample preparation. 2.5. Confocal laser scanning microscopy Confocal laser scanning microscopy (CLSM) was carried out with a Bio-Rad MRC 600 confocal laser scanning unit (Hemel Hempstead, UK) combined with an Leitz Ortholux 2 microscope (Heidelburg, Germany) having a 60 oilimmersion objective lens. A 0.1 vol% solution of Rhodamine B (Sigma Chemicals) was used to stain for the caseinate. The excitation source (488 nm) was an air-cooled Ar/Kr laser. The emulsion sample was placed in a welled slide with a coverslip. Confocal images were recorded 24 h after homogenization.

2. Materials and methods 2.1. Materials Spray-dried sodium caseinate (5.2 wt% moisture, 0.05 wt% calcium) was obtained from DeMelkindustrie (Veghel, The Netherlands). The 20 mM imidazole buffer (Sigma Chemicals, 99.0%) was prepared using distilled water and adjusted to pH 6.8 using hydrochloric acid (Fisher Chemicals, 37%); 0.01 wt% sodium azide was added to the buffer as an anti-microbial agent. Calcium chloride 2hydrate (147.0 g mol21, 99.5%) and n-tetradecane (99.0%) were supplied by Sigma Chemicals, as was the surfactant Tween 20, i.e. polyoxyethylenesorbitan monolaurate containing # 50% lauric acid (1.23 kDa). Calcium chloride was kept in a desiccator to prevent ingress of moisture. 2.2. Emulsion preparation Samples of oil-in-water emulsions (30 vol% n-tetradecane) were prepared using a laboratory-scale jet homogenizer (developed in the Procter Department of Food Science) operating in the pressure range 200 400 bar. Sodium caseinate was the sole emulsifying agent during homogenization. A notional molecular weight of 23 kDa was assumed for the caseinate in calculating the calcium/caseinate molar ratio R. Calcium chloride (R in the range 0 10) was dissolved in the aqueous phase prior to homogenization. Droplet-size distributions were measured using a Mastersizer 2000 multi-angle static light-scattering instrument (Malvern Instruments, UK). The average volume-surface droplet diameter of each freshly prepared emulsion was d32 0.4 ^ 0.05 mm with the exception only of the high calcium-content emulsions (R $ 8). About 30 min after homogenization, Tween 20 (0 2 wt%, 0 9 molar ratio with caseinate) was added to the emulsion with moderate stirring to ensure uniform distribution of the surfactant.

3. Results On the basis of visible creaming and particle-size distribution data assessed after 48 h, a stability diagram was constructed for sets of caseinate-stabilized n-tetradecanein-water emulsions (4 wt% protein, 30 vol% oil) with compositions varying with respect to concentrations of calcium chloride and Tween 20. This diagram is shown in Fig. 1, where the two axes correspond to the CaCl2 concentration, expressed as calcium/caseinate molar ratio R, and the concentration (in wt%) of Tween 20 added after homogenization. For all the compositions indicated in Fig. 1, except for the high ionic calcium systems (R $ 8),

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Fig. 1. Stability diagram for caseinate-stabilized oil-in-water emulsions (30 vol% n-tetradecane, 4 wt% protein, pH 6.8) with various compositions of ionic calcium and non-ionic surfactant stored for 48 h at 25 8C. Filled symbols (,P,O) denote stable emulsions (no visible serum separation). Open symbols (W,L,K) denote unstable emulsions (visible serum separation). Labels I and II denote primary and secondary stability regions, respectively. Specic symbol meanings: W, depletion occulation mainly from excess caseinate; K, depletion occulation mainly from excess Tween 20; L, calcium-induced electrostatic/bridging occulation; , stabilized with respect to occulation mainly by ionic calcium; O, stabilized with respect to occulation by low concentrations of both ionic calcium and nonionic surfactant; P, stabilized with respect to occulation by high concentrations of both ionic calcium and non-ionic surfactant; , intermediate boundary conditions.

Fig. 2. Rheology of a series of surfactant-free caseinate-stabilized emulsions (30 vol% oil, 4 wt% protein, pH 6.8, 25 8C). The loglog plot of apparent viscosity h against shear stress t is shown for different values of the calcium/caseinate molar ratio R: W, 0; , 3; K, 5; O, 8; L, 10.

the emulsions possess a uniform volume surface mean diameter (d32 0.40 ^ 0.05 mm). Filled symbols denote stable emulsions with no obvious creaming after 2 days and a steady monomodal particle-size distribution. It can be seen that there are two distinct islands of stability (labelled I and II) surrounded by regions of instability (open symbols). Let us rst consider the surfactant-free system compositions corresponding to points along the ordinate axis in Fig. 1. For low calcium molar ratios (0 # R # 4), the emulsions are monomodal in particle-size distribution, but unstable with respect creaming due to depletion occulation by excess protein in the continuous phase in the form of caseinate sub-micelles (5 10 nm), as described previously (Dickinson & Golding, 1997a; Dickinson et al., 1997). But at higher calcium ion contents, corresponding to R values in the range 5 7, the emulsion samples remain stable towards creaming over the 2 day observational period as result of the inhibition of depletion occulation by addition of ionic calcium (Dickinson & Golding, 1998a; Dickinson et al., 2001). With further increase in Ca2 concentration (R $ 8), instability again emerges through calcium-induced bridging occulation, as conrmed by the bimodal particle-size distribution determined by light scattering and the precipitated gel appearance of the emulsion after homogenization. The emulsion with R 8 is given intermediate stability status in Fig. 1, because it was found that some samples having this level of ionic calcium initially exhibited a bimodal particle-size distribution, although they reverted to monomodal character some 12 h after homogenization.

Rheological properties of a set of surfactant-free emulsions with R in the range 0 10 are presented in Fig. 2. We now consider surfactant-containing systems. Results for compositions along the abscissa in Fig. 1, corresponding to completely calcium-free emulsions, are consistent with the trend of synergistic depletion occulation behaviour of caseinate Tween 20 systems reported previously (Dickinson et al., 1999). With regards to the effect of surfactant on systems with low and intermediate concentrations of added CaCl2, the stability diagram shows that Tween 20 addition up to 0.5 wt% leads to a broadening of stability region I. Further increase in the surfactant content (. 0.75 wt%) leads to instability from surfactant-induced depletion occulation. That is, the surfactant is at a sufciently high overall concentration to displace most of the caseinate from the oil water interface, thereby increasing the number density of depleting species in the continuous phase (caseinate sub-micelles, Tween 20 micelles, and possibly also some mixed micellar aggregates). The inuence of surfactant content (0 2 wt% Tween 20) on the rheology of some emulsions of intermediate ionic calcium content R 5 is shown in Fig. 3. In certain parts of the composition space, the addition of surfactant causes behaviour of additional complexity. For instance, a stable emulsion (i.e. no creaming within 48 h) can be formed by adding a small quantity of Tween 20 (0.25 0.75 wt%) to a previously unstable emulsion (exhibiting depletion occulation from excess caseinate), provided that there is low content of ionic calcium present (2 # R # 4). This effect is represented in Fig. 1 as the triangular peninsula forming the lower part of stability region I. Fig. 4 gives a rheological characterization of these emulsion compositions (R 3; 0 0.875 wt% Tween 20) that cross this unstable/stable/unstable pathway. The secondary stability region II in Fig. 1 is brought about by the addition of relatively high concentrations of

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Fig. 5. Particle-size distribution P(d) for three caseinate-stabilized emulsions (30 vol% oil, 4 wt% protein, pH 6.8, 25 8C) of different compositions: A, R 5; 0 wt% Tween 20; B, R 10; 0 wt% Tween 20; C, R 10; 1 wt% Tween 20.

Fig. 3. Rheology of a series of caseinate-stabilized emulsions (30 vol% oil, 4 wt% protein, pH 6.8, 25 8C) containing a xed concentration of ionic calcium R 5: The loglog plot of apparent viscosity h against shear stress t is shown for different Tween 20 concentrations (wt%): W, 0; , 0.5; K, 0.75; O, 1; L, 2.

Tween 20 ($ 1 wt%) to calcium-rich occulated emulsions (R $ 8). In fact, since the apparent emulsion stability of region II is dened differently from that of region I, it might better be called a pseudo-stable region. This is because the gelled state at these high R values prevents the use of visual creaming as a test for emulsion stability; so particle-size distribution data have to be relied upon instead. Initially the emulsions (gels) were considered unstable with respect to calcium-induced bridging occulation on the basis of their showing a bimodal distribution in the Mastersizer (with or without intensive stirring). A stable monomodal emulsion was produced with the addition of 1 wt% Tween 20 (see Fig. 5). In such cases a moderate degree of stirring was required in order to distribute the surfactant uniformly

throughout the sample; this led to visible irreversible breakdown of the gelled structure. Region II in Fig. 1 is of narrow extent with respect to the surfactant concentration, with surfactant-induced depletion occulation occurring at $ 1.25 wt% Tween 20. The triangular shape of region II indicates that, with increasing R, more Tween 20 is required to generate unstable conditions (surfactant-induced depletion occulation). The location of the boundaries of region II was found to be sensitive to the age of the samples. So, for instance, systems with compositions in the ranges R 8 9 and 0.75 0.875 wt% Tween 20 were also found to exhibit phase separation on storage for signicantly longer than 48 h. Let us now consider the emulsion rheology in more detail. A characteristic feature of a occulated emulsion is its shear-thinning rheology (Barnes, 1994; Dickinson, 1998). This can be represented empirically by a powerlaw model,

t kDn ;

Fig. 4. Rheology of a series of caseinate-stabilized emulsions (30 vol% oil, 4 wt% protein, pH 6.8, 25 8C) containing a xed concentration of ionic calcium R 3: The loglog plot of apparent viscosity h against shear stress t is shown for different Tween 20 concentrations (wt%): W, 0; , 0.25; K, 0.5; O, 0.875.

where t is the shear stress, D is the shear-rate, k is the consistency index, and n is the power-law index. For a Newtonian liquid, we have n 1: Shear-thinning behaviour is indicated by n 21 . 1. The degree of t of individual experimental data curves to the power-law model was found to be very good (R 2 $ 0.97). Values of the power-law index for different curves obtained for the same emulsion composition were found to be reproducible to within 5 10%. Fig. 2 shows a log log plot of shear viscosity h versus stress t for a set of surfactant-free caseinate-stabilized emulsions (4 wt% protein, 30 vol% oil) with different concentrations of ionic calcium (0 # R # 10). The occulated emulsion with no added CaCl2 R 0 has a relatively high, low-stress viscosity (h , 40 Pa s). This can be attributed (Dickinson & Golding, 1997a,b) to entrapment of continuous phase within the aggregated structure increasing the effective volume fraction of hydrodynamically interacting entities. The sample exhibits shearthinning behaviour n21 2:2 as a result of the release of trapped continuous phase from the occulated structure, which is gradually broken down with increasing shear

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stress. Raising the Ca2 content causes a reduction in the low-stress viscosity down to a minimum value of h , 0.4 Pa s for the emulsion with R 5: This emulsion, which has considerably less pronounced shear-thinning behaviour n21 1:7; lies in the stable region I of Fig. 1. With a further increase in Ca2 content to R 8; the lowstress viscosity increases again and the system becomes signicantly more non-Newtonian n21 2:2; consistent with the onset of calcium-induced bridging occulation. The emulsion sample containing the highest CaCl2 concentration R 10 exists in a highly aggregated gel state that is not broken down signicantly over the shear stress range investigated. Apart from the high Ca2 content systems (R $ 8), the ow curves in Fig. 2 are characterized by reversibility with respect to ageing and applied stress. This behaviour is characteristic of systems that are weakly occulated by depletion interactions (Dickinson & Golding, 1997a,b). Fig. 3 shows how the concentration of added Tween 20 affects the rheology of a set of caseinate-stabilized emulsions (4 wt% protein, 30 vol% oil) containing the same content of added ionic calcium R 5: The surfactant-free system shows slight shear-thinning behaviour as already described. Remaining within the stable region I of Fig. 1, on addition of a moderately low level of Tween 20 (0.50 or 0.75 wt%), the emulsion becomes essentially Newtonian (n 21 , 1.1) with a very low viscosity (h , 0.01 Pa s). A further increase in Tween 20 concentration to 1 wt%, however, leads to a jump of almost three orders of magnitude in the low-stress viscosity (h , 0.7 Pa s) and the onset of substantial shear-thinning behaviour n21 2:1: This is consistent with the emulsion becoming destabilized as a result of surfactant-induced depletion occulation. All the ow curves displayed in Fig. 3 are reversible with respect to the shear conditions. Rheological properties for emulsion compositions of constant ionic calcium content R 3 around the peninsula of stable region I are shown in Fig. 4. The surfactantfree unstable emulsion has a moderate low-stress viscosity (h , 0.5 Pa s) and shear-thinning character n21 2:1: Addition of a small quantity of Tween 20 (0.25 or 0.5 wt%) causes a progressive reduction in low-stress viscosity h (to 0.25 or 0.07 Pa) and in the value of the inverse power-law index n 21 (to 1.6 or 1.3), which is consistent with a reduction in the degree of occulation. Pure Newtonian rheology is not observed, suggesting that there may still be some localized ocs present, but clearly not sufcient to cause signicantly enhanced global creaming, since these compositions form part of stable region I. However, a further increase in Tween 20 concentration to 0.875 wt% displaces the behaviour back into the unstable region, with distinctly shear-thinning rheology (h , 0.6 Pa, n21 2:1). Again all the ow curves in Fig. 4 are reversible with respect to the shear conditions. The ability of the particle-size distribution analysis to detect instability arising from non-reversible occulation is

illustrated in Fig. 5. Curve A is a typical monomodal distribution for the composition R 5; 0 wt% Tween 20, which is found in the stable region I of Fig. 1. On addition of excess CaCl2 (to give R 10) the resulting surfactant-free emulsion changes to a bimodal particle-size distribution (curve B) as a result of calcium-induced bridging occulation (Dickinson, Whyman, & Dalgliesh, 1987). Also shown in Fig. 5 is a nearly monomodal particle-size distribution (curve C) for an emulsion composition (R 10; 1 wt% Tween 20) found in stable region II of Fig. 1. The slight tail in curve C (Fig. 5) may be due to some lingering calcium-mediated bridging ocs of predominantly surfactantcoated droplets, or to a small fraction of larger droplets arising as a result of less efcient homogenization in the presence of caseinate aggregated by calcium binding/ bridging. A relatively high Tween 20 concentration (1 wt%), then yields a monomodal size distribution when the surfactant is added to a visibly precipitated emulsion gel exhibiting a bimodal size distribution. Genuine depletionocculated samples characteristically yield a monomodal distribution on examination by light scattering, due to the easy reversibility of this kind of weak occulation towards sample dilution (Dickinson & Golding, 1997a). In order to conrm these inferences from rheology and creaming about the effects of ionic calcium and non-ionic surfactant on the state of occulation of the emulsions (4 wt% caseinate, 30 vol% oil), selected samples of different compositions within the stability diagram (Fig. 1) were examined by CLSM. The caseinate was labelled with Rhodamine B. Micrographs of three such samples are presented in Fig. 6. Picture A is for the occulated reference system in the absence of added CaCl2 or Tween 20, i.e. a composition corresponding to the bottom left corner of the stability diagram. The intense white network corresponds to interconnected regions of protein-coated emulsion droplets that are clustered together presumably under the inuence of depletion interactions. Fig. 6(A) resembles the sort of image published by Srinivasan et al. (2001). Confocal micrograph B in Fig. 6 is for a sample with R 4:5 and no added surfactant. The composition lies on the border of stability region I. Numerous (white) proteincoated droplet clusters of limited size can be seen, with a (dark) uid-like background of individual emulsion droplets. With slight increase in Ca2 content (to R $ 5) the entire eld of view becomes devoid of these clusters, with an appearance (not shown) similar to the mobile dark background. Thus, we can be sure that Fig. 6B represents an intermediate state of emulsion microstructure and stability on the boundary of region I between fully stable and clearly occulated states. Interestingly, at this transitional composition of limited Ca2 concentration R 4:5; the emulsions do not appear by eye to cream, even though some limited depletion occulation is evident under the microscope. Confocal micrograph C in Fig. 6 refers to a composition with R 5 and 2 wt% Tween 20. At this high surfactant

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Fig. 7. A stability diagram for emulsions (30 vol% oil, pH 6.8) of low caseinate content (2 wt% protein) with various compositions of ionic calcium and non-ionic surfactant stored for 48 h at 25 8C. Filled symbols () denote stable emulsions (no visible serum separation). Open symbols (L,K) denote unstable emulsions (visible serum separation). Specic symbol meanings: K, depletion occulation mainly from excess Tween 20; L, calcium-induced electrostatic/bridging occulation; , no occulation; , intermediate boundary condition.

Fig. 6. Confocal laser scanning micrographs for three caseinate-stabilized emulsions (30 vol% oil, 4 wt% protein, pH 6.8, 25 8C) of different compositions: A, R 0; 0 wt% Tween 20; B, R 4:5; 0 wt% Tween 20; C, R 5; 2 wt% Tween 20. Protein is stained with Rhodamine B. Scale bar 5 mm. In micrograph B, the arrows denote (i) lingering depletion ocs (light) and (ii) a stable background of discrete emulsion droplets (dark). In micrograph C, the arrows denote (i) phase-separated serum containing displaced protein (light) and (ii) surfactant-induced depletion ocs (dark).

concentration, all the protein is displaced from the oil water interface (Heertje, Nederlof, Hendrickx, & Lucassen-Reynders, 1990). In contrast to Fig. 6A, the white regions here represent protein-containing serum-rich areas, and the darker regions represent fractal-type aggregated networks of depletion occulated droplets. Some confocal micrographs (not shown) were obtained for emulsions that became strongly occulated in the

presence of a high ionic calcium concentration. The images exhibited a dense, white, immobile appearance with visible evidence of heavy cross-linking. This type of emulsion system became irreversibly broken down upon moderate shearing, to give the appearance of a stable, uid-like, emulsion containing many discrete entities similar to the background of Fig. 6B (see earlier). All the above experiments were carried out on ne emulsions (0.4 mm diameter) containing 4 wt% caseinate and 30 vol% n-tetradecane. Of course, we recognize that the precise form of the stability diagram might be expected to depend on the oil volume fraction, the average droplet size, and especially the protein/oil ratio. To investigate the sensitivity of the stability map to the protein/oil ratio, parallel experiments were carried out for 30 vol% emulsions containing different concentrations of protein in the range 1 4 wt%. Following Fig. 1 with regard to the axis labelling conventions, Fig. 7 shows the stability diagram for a set of emulsions containing 2 wt% caseinate. In the absence of added Ca2 or surfactant, this emulsion has good stability with respect to occulation because of the combination of a fully saturated adsorbed layer and little unadsorbed protein in the continuous phase (Dickinson et al., 1997). Hence, at low surfactant concentrations, the primary stability region I in Fig. 7 is broader in terms of the range of R values than it is in Fig. 1 for the 4 wt% caseinate system. In contrast, so long as the mean droplet size is roughly the same for all the stable emulsions (d32 0.40 ^ 0.05 mm), one should not expect the absolute concentration of caseinate to affect signicantly the value of R required to induce calcium-induced bridging occulation. This is indeed what is found, with the upper boundary of region I located similarly (at R < 7) in Figs. 1 and 7. Furthermore, surfactant-led depletion occulation

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occurs in Fig. 7 at a composition just above 0.75 wt% Tween 20, which is also in keeping with the behaviour depicted in Fig. 1. Hence, it appears that the effect of caseinate/oil ratio on the position of the primary stability region I can be well understood in terms of existing knowledge.

4. Discussion This investigation conrms previous observations (Dickinson & Golding, 1998a; Ye & Singh, 2001) that reversible depletion occulation of caseinate-stabilized emulsions containing excess protein is inhibited by addition of a concentration of calcium ions well below that required to precipitate the protein. The Ca2 concentration required to inhibit creaming of the emulsions studied here, containing 4 wt% caseinate and 30 vol% oil, is rather higher (ca. 8 mM) than that determined previously (ca. 5 mM) by Dickinson and Golding (1998a,b). Furthermore, in this study the rheology of the calcium-inhibited depletion emulsion system is still slightly shear-thinning, in comparison with the more nearly Newtonian behaviour reported by Dickinson and Golding (1998a,b). Such differences may be attributable to differences in chemical composition or degree of degradation or polymerization of the caseinate sample, and/or differences in homogenization conditions and procedures employed in the two investigations. The composition determined here for the onset of calcium-induced bridging occulation (R , 8, [Ca2], 14 mM) falls consistently within the ionic calcium content range of 12 ^ 2 mM reported previously for this type of calcium-induced aggregation (Dickinson & Davies, 1999; Dickinson & Golding, 1998a). High ionic calcium concentrations may generate calcium-induced occulation by a combination of factorsreducing the net charge on the adsorbed protein, compacting the adsorbed layer and thereby reducing the steric repulsion, or screening charges between layers and thereby diminishing the electrical double-layer repulsion. Some calcium-containing emulsions in the stability transition zone R 8 are reported here to exhibit reversible changes in particle-size distribution with time, i.e. reverting from an initial bimodal distribution to a monomodal one after ca. 12 h of storage. This may indicate some structural rearrangement within the adsorbed caseinate layer, or a redistribution of bound calcium ions with time as the system relaxes following the intense local forces imposed during homogenization. Considering now our new ndings for systems containing both ionic calcium and non-ionic surfactant, the main general conclusion is that the behaviour of the global stability diagram (Fig. 1) is largely explicable in terms of identiable contributions from the two added species. One interesting observation is the creation of a stable emulsion from a occulated one containing a low level of CaCl2 R 2 4 by addition of a relatively small amount of Tween 20

(0.25 0.75 wt%). The likely explanation goes as follows. In a system of limited ionic calcium content R 2 4; a large proportion of it will be effectively removed from the aqueous phase as caseinate adsorbs at the oil water interface during homogenization. That is, there is too low a Ca2/caseinate ratio in the aqueous phase signicantly to increase the size or reduce the number density of caseinate sub-micelles between the droplets, and so depletion occulation still occurs in the system. However, the addition of a small quantity of Tween 20 will have the effect of competitively displacing much of the adsorbed protein and, crucially, its accompanying ionic calciumfrom the surface of the droplets. The increase in Ca2 concentration in the aqueous phasemainly not free but bound to nonadsorbed proteinwill lead to formation of larger caseinate sub-micelles and a consequent reduction in the number density of depleting species. This reduces the osmotic pressure driving force, which in turn makes the depletion interaction less thermodynamically favourable (Dickinson et al., 2001). Further addition of Tween 20 destabilizes the system again due to depletion occulation by surfactant micelles (or mixed surfactant/protein micelles). This progression from instability to stability to instability again with increasing surfactant concentration is mirrored by a clear trend in rheological behaviour (see Fig. 4) from shearthinning to (nearly) Newtonian to shear-thinning again. Another interesting feature of the global stability diagram concerns the observation that a relatively high calcium/caseinate ratio combined with a relatively high surfactant concentration brings about a secondary region of stability (region II in Fig. 1). By itself, a high level of Ca2 (R $ 8) causes calcium-induced bridging occulation. Through a competitive adsorption mechanism, addition of sufcient Tween 20 (1 wt%) with moderate stirring displaces most of the calcium-mediated caseinate bridges from the droplet surfaces, thereby disrupting the ocs, as indicated by the change to an essentially monomodal particle-size distribution. Stability is lost again, however, at higher Tween 20 concentrations ($ 1.25 wt%) due to putative depletion occulation by excess surfactant micelles. An additional factor relevant to the boundaries of region II is that calcium-induced cross-linking within the adsorbed casein layer may make protein competitive displacement by surfactant more difcult, as has been found in mixed protein systems (Hunt, Dickinson, & Horne, 1993). While the oil content in this study was kept constant (30 vol%), in order to focus attention on other compositional variables, the dispersed phase volume fraction is generally regarded as a signicant factor affecting the creaming stability of a caseinate-based emulsion (Dickinson et al., 1997). Nevertheless, we expect the qualitative stability features of this mixed system to be independent of oil volume fraction. Regarding quantitative aspects, we can envisage that the stability region I will increase in area with increase in oil volume fraction, as a greater dispersed

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phase fraction contributes a greater emulsion viscosity and hence more slowing down of gravity-driven phase separation processes. On lowering the oil content, this same stable region might be expected to shrink and shift position towards higher calcium ion concentrations, though it is not impossible that a synergistic competitive effect of surfactant and (calcium) caseinate, as highlighted above, could provide further local regions of stability in composition space.

5. Conclusions It is conrmed that the presence of ionic calcium (8 mM) in emulsions containing a high concentration of sodium caseinate inhibits depletion occulation. The further addition of non-ionic surfactant has been found to have a synergistic effect in promoting conditions of stability on both sides of this window of calcium-inhibited depletion occulation (8 14.5 mM [Ca2]). Two specic types of behaviour are noteworthy. (1) At low calcium ion concentrations, surfactant-free emulsions are occulated by excess caseinate in the continuous phase, but the addition of a small quantity of Tween 20 (0.25 0.75 wt%) generates emulsions that are stable to creaming (over 48 h). (2) In the case of strongly occulated emulsions containing a fairly high concentration of ionic calcium, the addition of a substantial amount of Tween 20 (1 wt%) produces stable emulsions characterized by monomodal particle-size distributions based on light scattering. Both of these phenomena rely on competitive displacement of protein from the oil water interface by Tween 20. This displacement has the effect of (i) increasing the effective calcium ion concentration in the continuous phase available for interacting with non-adsorbed sub-micellar caseinate at low calcium/caseinate molar ratios, and (ii) disrupting the calcium-mediated bridges between occulated droplets at high calcium/caseinate molar ratios. Acknowledgments S.J.R. acknowledges receipt of a BBSRC CASE Studentship in collaboration with Unilever Research (Colworth Laboratory), at whose premises the confocal microscopy was carried out. References
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